Automated molecular pathology apparatus having independent slide heaters

ABSTRACT

Apparatus and methods for automatically staining or treating multiple tissue samples mounted on microscope slides are provided. Individualized slide temperature control is accomplished by the heating system according to the present invention that has thermal platforms radially mounted to the carousel for heating the slides and sensing the temperature of each. The heating system also permits automated de-waxing if necessary.

CROSS-REFERENCE APPLICATION

[0001] This is a continuation-in-part application of U.S. applicationSer. No. 09/259,240 filed on Feb. 26, 1999, the subject matter of whichis hereby incorporated herein. This application is also acontinuation-in part of U.S. Application Serial No. 60/076,198 filed onFeb. 27, 1998, the subject matter of which is hereby incorporatedherein.

FIELD OF THE INVENTION

[0002] The present invention is directed to apparatus for use indiagnostic molecular pathology and, more particularly, to such apparatusused for the automated staining and/or treating of tissue samplesmounted on microscope slides.

BACKGROUND OF THE INVENTION

[0003] Molecular pathology is the examination at a molecular level ofthe DNA, mRNA, and proteins that cause or are otherwise associated withdisease. From this examination important information about patientdiagnosis, prognosis, and treatment options can be elucidated. Thepractice of molecular pathology is generally divided into two mainareas: (i) analysis of DNA, mRNA, and proteins in intact cells(in-situ), and (ii) analysis of these biological materials after theyhave been extracted from tissues. The first category, to which thepresent invention is primarily directed, has the advantage that itallows the pathologist or scientist to study the histopathologicarchitecture or morphology of the tissue specimen under the microscopeat the same time that the nucleic acid or proteins are being assayed.These techniques include immunohistochemistry (IHC) which looks atproteins, in-situ hybridization (ISH) which looks at nucleic acids,histochemistry (HC) which looks at carbohydrates, and enzymehistochemistry (EHC) which looks at enzyme chemistry. For example, ISHcan be used to look for the presence of a genetic abnormality orcondition such as amplification of cancer causing genes specifically incells that, when viewed under a microscope, morphologically appear to bemalignant. ISH is also useful in the diagnosis of infectious diseases asit allows detection not only of a microbial sequence but also ofprecisely which cells are infected. This may have importantclinicopathologic implications and is an effective means to rule out thepossibility that positive hybridization signal may have come from anadjacent tissue of no clinical concern or from blood or outsidecontamination.

[0004] IHC utilizes antibodies which bind specifically with uniqueepitopes present only in certain types of diseased cellular tissue. IHCrequires a series of treatment steps conducted on a tissue section orcells (e.g. blood or bone marrow) mounted on a glass slide to highlightby selective staining certain morphological indicators of diseasestates. Typical steps include pretreatment of the tissue section toremove the paraffin and reduce non-specific binding, retrieval ofantigens masked by cross-linking of the proteins from the chemicalfixatives, antibody treatment and incubation, enzyme labeled secondaryantibody treatment and incubation, substrate reaction with the enzyme toproduce a fluorophore or chromophore highlighting areas of the tissuesection having epitopes binding with the antibody, counterstaining, andthe like. Most of these steps are separated by multiple rinse steps toremove unreacted residual reagent from the prior step. Incubations canbe conducted at elevated temperatures, usually around 37° C., and thetissue must be continuously protected from dehydration. ISH analysis,which relies upon the specific binding affinity of probes with unique orrepetitive nucleotide sequences from the cells of tissue samples orbodily fluids, requires a similar series of process steps with manydifferent reagents and is further complicated by varying temperaturerequirements.

[0005] In view of the large number of repetitive treatment steps neededfor both IHC and ISH, automated systems have been introduced to reducehuman labor and the costs and error rate associated therewith, and tointroduce uniformity. Examples of automated systems that have beensuccessfully employed include the NEXES® and Gen II® staining Systemsavailable from Ventana Medical Systems (Tucson, Ariz.) as well as thesystem disclosed in U.S. Pat. No. 5,654,199 to Copeland et al. Thesesystems employ a microprocessor controlled system including a revolvingcarousel supporting radially positioned slides. A stepper motor rotatesthe carousel placing each slide under one of a series of reagentdispensers positioned above the slides. Bar codes on the slides andreagent dispensers permits the computer controlled positioning of thedispensers and slides so that different reagent treatments can beperformed for each of the various tissue samples by appropriateprogramming of the computer.

[0006] The aforementioned staining systems include either a hot airblower or a heat lamp to warm the samples above laboratory ambienttemperatures for steps requiring elevated temperatures. Heating theslide improves staining quality by acceleration of the chemical reactionand can permit a reaction temperature more closely matching bodytemperature (about 37° C.) at which antibodies are designed to react.While such convection or radiant heating systems have been generallysuitable for IHC, which is antibody based, they are less suitable forISH, which is nucleic acid based and requires higher and more precisetemperature control. In order to denature the DNA double helix of boththe target sample and the probe so as to render them single stranded,the temperature must be raised above the melting point of the duplex,usually about 94° C. At the same time it is imperative that the samplenot be overheated past 100° C. as doing so destroys cell morphologymaking it difficult to view under a microscope. Precise temperaturecontrol is also required in ISH to effect probe hybridization at thedesired stringency. The selected temperature must be low enough toenable hybridization between probe and template, but high enough toprevent mismatched hybrids from forming. It would be desirable,therefore, to have an automatic tissue staining apparatus which cancontrol the temperature of reactions with enough precision for most ISHapplications.

[0007] Another disadvantage of the heating units typically employed withautomated tissue stainers is that they do not permit the temperature ofindividual slides to be separately controlled. With prior art systemsall of the slides are heated to the same temperature at any given timeduring the process. For example, U.S. Pat. No. 5,645,114 to Bogen et al.discloses a dispensing assembly adapted to carry a plurality ofmicroscope slides. Individual slide holders containing resistive heatingunits are provided. However, with the assembly taught by Bogen et al.,all of the slides would be heated to a common temperature because, forexample, no means are disclosed for separate heating controls or forshielding slides from heat generated by adjacent slides. This precludesprotocols having different temperature parameters from being run ondifferent samples at the same time. For example, DNA probe assays havingdifferent stringency requirements could not be run efficiently at thesame time. It would be desirable, therefore, to have an automatic tissuestaining apparatus wherein adjacent slides can have different testsapplied to them even when the tests have unique heating requirements.

[0008] A difficulty frequently encountered in both IHC and ISH testingresults from the manner in which the tissues are typically preserved.The mainstay of the diagnostic pathology laboratory has been for manydecades the formalin-fixed, paraffin embedded block of tissue, sectionedand mounted upon glass slides. Fixation in such a preservative causescross-linking of macromolecules, both amino acids and nucleic acids.These cross-linked components must be removed to allow access of theprobe to the target nucleic acid and to allow the antibody to recognizethe corresponding antigen. “Unmasking” the antigen and/or nucleic acidis typically accomplished manually with multiple pretreatment,protolytic digestion, and wash steps. It would be desirable if theprocess of conditioning cells so that their antigens and nucleic acidsare available for detection could be automated.

[0009] Prior to staining, complete removal of the paraffin is alsorequired so that it does not interfere with antibody or probe binding.Paraffin, a hydrophobic substance, must be removed prior to staining orhybridization using probes. Deparaffinization is normally achieved bythe use of two or three successive clearing reagents that are paraffinsolvents such as xylene, xylene substitutes or toluene which may betoxic, flammable and pose environmental hazards. Safer and fastermethods to deparaffinize the slides would be advantageous.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to apparatus and methods forautomatically staining or treating multiple tissue samples mounted onmicroscope slides so that each sample can receive an individualizedstaining or treatment protocol even when such protocols requiredifferent temperature parameters. Thus, different DNA probe and/orantibody based staining procedures can be run simultaneously despite thefact that each may have different heating requirements at a given pointin time. Additionally, samples requiring de-waxing (e.g. tumor sections)can be automatically processed at the same time as other samples (e.g.smears) that do not require this preliminary step.

[0011] More specifically, the apparatus is a computer controlled, barcode driven, staining instrument that automatically applies chemical andbiological reagents to tissue or cells mounted or affixed to standardglass microscope slides. Up to 20 slides are mounted in a circular arrayto a carousel which rotates, as directed by the computer, placing eachslide under one of a series of reagent dispensers positioned above theslides. Each slide receives the selected reagents (e.g. DNA probe) andis washed, mixed and/or heated in an optimum sequence and for therequired period of time. Tissue sections so stained or treated are thenremoved from the apparatus by the user to be viewed under a microscopeby a medical practitioner who reads the slide for purposes of patientdiagnosis, prognosis, or treatment selection. The computer controlledautomation permits use of the apparatus in a “walk-away” manner, i.e.with little manual labor.

[0012] Individualized slide temperature control is accomplished by theheating system according to the present invention that has thermalplatforms radially mounted to the carousel for heating the slides andsensing the temperature of each. A printed circuit board, also mountedto the slide carousel, individually monitors and controls each thermalplatform separately. Information and power pass between the rotatingcarousel and the fixed apparatus via a slip ring assembly. Thisinformation includes the upper and lower temperature parameters neededfor heating each of the 20 slides for the appropriate time period.

[0013] A key advantage of the present invention is that each sample canreceive an individualized staining or treatment protocol even when suchprotocols require different temperature parameters.

[0014] Another advantage of the present invention is that it allows thetemperature of the entire surface of the slide to which the tissue ismounted to be carefully controlled (i.e. within plus or minus twodegrees Celsius of the desired temperature). Such precision isparticularly necessary for DNA denaturation and probe hybridization inISH and related processes such as in-situ PCR. Furthermore, since theheating according to the present invention is made uniform, this narrowtemperature range is maintained throughout the surface of the slide sothat the tissue is evenly heated regardless of its position on theslide.

[0015] Still another advantage of the present invention is that itpermits the de-waxing of the tissue sample in an automated formatwithout reliance upon harmful solvents such as xylene.

[0016] Yet another advantage of the present invention is that by heatingthe tissue in an aqueous solution the tissue is better conditioned forstaining by rendering the targeted molecules in the cells moreaccessible to the stain.

[0017] Still another advantage of the present invention is its abilityto rapidly heat the surface of the slide to which the tissue is mounted(i.e. from 37° C. to 95° C. in under two minutes and to cool down oversame range in under four minutes) so as to permit DNA denaturationwithout over-denaturation and loss of cell morphology due to excessheating.

[0018] Yet another advantage of the present invention is its ability todehydrate the tissue sample following staining using heat.

[0019] Still another advantage of the present invention is the negationof human error and increase in productivity resulting from automation.

[0020] With the foregoing and other objects, advantages and features ofthe invention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the invention, the appended claims and to theseveral views illustrated in the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is a perspective view of the present invention shown withthe slide hood open and the carousel door removed.

[0022]FIG. 2 is a perspective view of the present invention shown inconjunction with a computer and other instruments with which itoperates.

[0023]FIG. 3 is an exploded view of the present invention.

[0024]FIG. 4 is a perspective view of the present invention shown with areagent dispenser.

[0025]FIG. 5 is a block diagram of the heating system of the presentinvention.

[0026]FIG. 6 is a perspective view of the heater/sensor unit of thepresent invention shown with the plate partially broken away to revealthe pins which are part of the control electronics.

[0027]FIG. 7 is a plan view of a heater.

[0028]FIG. 8 is a perspective view of a thermal platform shown with aglass slide thereon.

[0029]FIG. 9 is a cross section view of the thermal platform taken alongline 9-9 of FIG. 8.

[0030]FIG. 10 is a perspective view of the slip ring assembly and slidecarousel shown with a plurality of thermal platforms mounted thereto.

[0031]FIG. 11 is a perspective view of the underside of the carouselshown in FIG. 10 shown with the control electronics printed circuitboard mounted thereto.

[0032]FIG. 12 is a top plan view of the heater;

[0033]FIG. 13 is a block diagram of the control electronics, heaters andsensors as shown in FIG. 5.

[0034]FIG. 14 is a flow chart of the controlling of the heating of anindividual slide.

[0035]FIG. 15 is a flow chart of the controlling of the cooling of anindividual slide.

[0036]FIG. 16 is a cross section view of the ring assembly.

[0037]FIG. 17 illustrates the results obtained for PCR amplificationsperformed using the apparatus of the present invention and usingstandard techniques; lane 0: size marker, lane 1: standard PCR withhapten-dNTP mix, lane 2: standard PCR with dNTP mix, lane 3: on-slidePCR with hapten-dNTP mix, lane 4: on-slide PCR with dNTP mix;

[0038] FIGS. 18A-18B illustrate the results obtained following on-slidePCR amplification and on-slide antibody hybridization.

DETAILED DESCRIPTION OF THE INVENTION

[0039] Referring now in detail to the drawings wherein like parts aredesignated by like reference numerals throughout, there is illustratedin FIG. 1 a perspective view of the molecular pathology apparatusaccording to the present invention which is designated generally byreference numeral 10. Apparatus 10 is designed to automatically stain orotherwise treat tissue mounted on microscope slides with nucleic acidprobes, antibodies, and/or reagents associated therewith in the desiredsequence, time and temperature. Tissue sections so stained or treatedare then to be viewed under a microscope by a medical practitioner whoreads the slide for purposes of patient diagnosis, prognosis, ortreatment selection.

[0040] In a preferred embodiment, apparatus 10 functions as onecomponent or module of a system 12 (FIG. 2) which also comprises a hostcomputer 14 preferably a personal computer, monitor 16, keyboard 18,mouse 20, bulk fluid containers 22, waste container 23 and relatedequipment. Additional staining modules or other instruments may be addedto system 12 to form a network with computer 14 functioning as a server.Alternatively, some or all of these separate components could beincorporated into apparatus 10 making it a stand-alone instrument.

[0041] The preferred configuration of apparatus 10 as well as system 12is generally as described in U.S. patent application Ser. No. 08/995,052filed Dec. 19, 1997 as well as in the Ventana NexES User 's Guideavailable from Ventana Medical Systems, Inc. (Tuscon, Ariz.), bothincorporated herein, except with respect to the novel heating system,slide support, bulk fluids module, volume adjust, and slide wipe asdisclosed below. For purposes of clarity, detailed descriptions of thosecomponents found in both the present invention and the incorporatedreferences are omitted.

[0042] In brief, apparatus 10 is a microprocessor controlled staininginstrument that automatically applies chemical and biological reagentsto tissue mounted on standard glass microscope slides. A carouselsupporting radially positioned glass slides is revolved by a steppermotor to place each slide under one of a series of reagent dispensers.Apparatus 10 controls dispensing, washing, mixing, and heating tooptimize reaction kinetics. The computer controlled automation permitsuse of apparatus 10 in a walk-away manner, i.e. with little manuallabor.

[0043] More particularly, apparatus 10 comprises a housing formed of alower section 30 removably mounted or hinged to an upper section 32. Aslide carousel 34 is mounted within lower section 30 for rotation aboutaxis A-A. As set forth in greater detail below, a plurality of thermalplatforms 50 are mounted radially about the perimeter of carousel 34upon which standard glass slides with tissue samples may be placed.Carousel 34 is preferably constructed of stainless steel. It is a keyfeature of the present invention that the temperature of each slide maybe individually controlled by means of various sensors andmicroprocessors as described herein. Also housed within apparatus 10(FIG. 3) are wash dispense nozzles 36, Coverslip™ dispense nozzle 37,fluid knife 38, wash volume adjust nozzle 39, bar code reader mirror 40,and air vortex mixers 42 the details of which are discussed hereinafter.

[0044] Rotatably mounted atop upper section 32 is a reagent carousel 28.Dispensers 26 are removably mounted to reagent tray 29 (FIG. 4) which,in turn, is adapted to engage carousel 28. Reagents may include anychemical or biological material conventionally applied to slidesincluding nucleic acid probes or primers, polymerase, primary andsecondary antibodies, digestion enzymes, pre-fixatives, post-fixatives,readout chemistry, and counterstains. Reagent dispensers 26 arepreferably bar code labeled 29 for identification by the computer. Foreach slide, a single reagent is applied and then incubated for a preciseperiod of time in a temperature-controlled environment. Mixing of thereagents is accomplished by compressed air jets 42 aimed along the edgeof the slide thus causing rotation of the reagent. After the appropriateincubation, the reagent is washed off the slide using nozzles 36. Thenthe remaining wash buffer volume is adjusted using the volume adjustnozzle 39. Coverslip™ solution, to inhibit evaporation, is then appliedto the slide via nozzle 37. Air knife 38 divides the pool of Coverslip™followed by the application of the next reagent. These steps arerepeated as the carousels turn until the protocol is completed.

[0045] In addition to host computer 14, apparatus 10 preferably includesits own microprocessor 44 to which information from host computer 14 isdownloaded. In particular, the computer downloads to microprocessor 44both the sequence of steps in a run program and the sensor monitoringand control logic called the “run rules” as well as the temperatureparameters of the protocol. Model No. DS2251T 128K from DallasSemiconductor, Dallas Tex. is an example of a microprocessor that canperform this function.

[0046] Turning now to FIG. 5 there is shown a block diagram of the slideheating system 48. The system generally comprises about twenty thermalplatforms 50, radially mounted to carousel 34, for heating the slidesand monitoring the temperature thereof, and control electronics printedcircuit board 52 also mounted to the slide carousel for monitoring thesensors and controlling the heaters. Control electronics 52 are mountedunder the rotating slide carousel 34. Information and power aretransferred from the fixed instrument platform to the rotating carouselvia a slip ring assembly 56. This information includes the temperatureparameters needed for heating the slides (upper and lower) communicatedfrom microprocessor 44 (after having been downloaded from computer 14)to control electronics 52 as described below. If, during a run, theslide temperature is determined to be below the programmed lower limit,the thermal platform heats the slide. Likewise, if the slide temperatureis found to be above the upper limit, heating is stopped. (See Block 88of FIG. 14). A power supply of sufficient capacity to provide abouteight watts per heater is provided to meet the requisite rate oftemperature rise (a/k/a “ramp up”).

[0047] Similarly, in an alternate embodiment, the cooling of the slidesmay be likewise controlled, as described subsequently. In one alternateembodiment, cooling platforms are mounted below the slide. The coolingplatforms may comprise Peltier-type thermal transducers. In analternative embodiment, a cooling device such as a fan (not shown) mayoptionally be provided if rapid cooling of the slides is required forparticular applications. The cooling device will modify the ambient airfor all of the platforms, necessitating the heaters corresponding to theslides which should not be cooled to compensate for the drop in ambientair temperature.

[0048] The slide heating system described herein uses conduction heatingand heats slides individually. The system provides more accurateon-slide temperature and allows for temperature settings on a slide byslide basis. Each of the components of heating system 48 are nowdescribed in more detail.

Thermal Platforms

[0049] With reference to FIGS. 6-10 thermal platforms 50 comprises twocomponents: a heater/sensor unit 58 and a housing 70 for removablymounting heater/sensor unit 58 to carousel 34 and for supporting theslides 37.

[0050] Heater/sensor unit 58 has a plate 60, about two inches long andone inch wide, preferably constructed of 0.04 inch thick brass plate. Inlieu of brass, another material may be employed if it is both rigid andhas sufficient conductivity to dissipate heat evenly throughout itssurface so that the slide is warmed in a uniform manner. The plate mayoptionally be covered with a corrosion resistant material such asTeflon®.

[0051] It is a particular feature of the present invention that adjacentslides may optionally be heated to different temperatures at particularpoints in time. This is accomplished by making the slides thermallyisolated from one another by having high thermal resistance between theheaters. As one skilled in the art will readily appreciate, thermalresistance is a function of the conductivity of the material, thethickness of the material, and the distance the heat must travel. Hence,a variety of materials may be employed to thermally isolate adjacentslides including rubber, plastics, ceramics, or metals if they providethermal resistance based on the aforementioned criteria. As used herein,the term “thermally isolated” means that the heat from one heater has noappreciable affect on the temperature of adjacent slides. In addition, avariety of structures may be employed to thermally isolate adjacentslides including mounting the slide on a thermally resistant material,mounting a thermally resistant material in between slides, and placingthe slides on a thermally resistant platform.

[0052] In a preferred embodiment, mounted about the perimeter of plate60 and depending perpendicularly from the underside thereof is a skirt62 preferably constructed of rubber or a similar material that is bothwaterproof and a thermal insulator. The insulative properties of skirt62 helps to facilitate these temperature differentials between adjacentslides. It should be appreciated that a variety of materials may beemployed in lieu of rubber to thermally insulate adjacent slidesincluding ceramics and plastics that can withstand temperatures of atleast 100° C. Also, the components housed within the cavity ofheater/sensor unit 58 (to be described) must be shielded from thevarious heated solutions (oil and water based) that will be appliedduring the staining operation of the apparatus. Hence, plate 60 ispreferably bonded to skirt 62 through vulcanizing or a similar processthat results in a fluid impervious seal even at high temperatures. Thewalls of skirt 62 must be similarly resistant to such heated solutions.In a preferred embodiment, the thickness of the skirt 62 is 0.06 inchfor rubber material, thereby minimizing the cross sectional area throughwhich heat may travel. Alternatively, skirt 62 may be constructed of ametallic material, such as brass, sufficiently thin (e.g., approximately0.01 inch) to provide thermal resistance. When mounted to carousel 34the height of skirt 62 is about one-half inch thereby elevating plate 60and the slide supported thereon by that same distance. Elevating theplatform upon which the slides are placed distances them from thevarious fluids that collect on the carousel 34 during operation andthermally isolate or insulate the slides from heat generated by adjacentheaters.

[0053] A photo-etched resistive heater 64 is mounted to the underside ofplate 60 within the cavity defined by skirt 62. The heater can be madeof a variety of materials from high resistance, nickel based materials,such as Inconel, to more conductive materials, such as cuppro-nickel.Cuppro-nickel is preferred. The photo-etched resistor 64 is bonded toand retained between two, thin sheets of plastic such 0.008 inch thickKapton™ polyimid film available from DuPont. Electrical leads 65 arewelded to the ends of the resistive traces and these leads are broughtout from between the two Kapton™ layers and connected to thephoto-etched resistor. One outer side of this sandwich is designated tobe bonded to the heater while the other outer side has another,photo-etched circuit attached to it. The second circuit is fortemperature sensing and has four low resistance traces which terminatein the center of the heater at solder pads which connect to a sensormade by Dallas Semiconductor, their part number DS1721 S. The fourtraces are welded to four wires which exit in the same area as the wiresconnected to the heating traces. A third layer of Kapton™ is bonded overthe control traces and this third layer has cutouts over the solder padsin the center so that the sensor can be attached. Six wires exit theheater, two for powering the heater and four for sensing thetemperature, as shown in FIG. 7. The six wires are attached to a female,six circuit plug 67 which could be made by a number of companies, suchas AMP (Harrisburg, Pa.) or Molex (Lisle, Ill.). The female connectorslides over six, mating pins 69 that are soldered onto the controlelectronics circular PC board 52 described elsewhere. The PC board 52 ismounted under the slide carousel 34 opposite the side upon which theslide heaters are attached, so the six pins extend upward through arectangular aperture 75 in the carousel.

[0054] A manufacturing source for heaters 64 according to thespecifications set forth herein is Minco Products (Minneapolis, Minn.).As set forth in more detail below, heaters may be individuallycontrolled by an integrated circuit driver or individual transistors(mounted to printed circuit board 52) capable of switching the heatercurrent on and off.

[0055] The heater/sensor unit as described has the ability to rapidlyheat the useful area of the slide from 37° C. to 95° C. in under twominutes and to cool down over same range in under four minutes so as topermit DNA denaturation without over-denaturation and loss of cellmorphology due to excess heating.

[0056] Heating the slide surface uniformly is another key goal of thepresent invention since tissue specimens are often mounted in differentpositions on the slide. This poses a challenge for conduction heating,even when done manually, since traditional hot plates often generatepatchy “hotspots” making it hard to know where to place the slide on theplate. If the cells throughout the tissue are not heated evenly, themicroscope slide cannot be accurately interpreted. For example, if thetemperature is not high enough to denature the probe and tissue DNA thismay lead to false negatives and inconsistencies during stringency washescould lead to false positives.

[0057] In order to ensure uniform heating by thermal platform 60, theresistive heater 64 is bonded to the bottom side of plate 60. As stated,the brass plate has sufficient conductivity to smooth out any localnon-uniformity caused by the fact that the heating traces are notcontinuous over the surface, but rather are adjacent lines separated bya space where no heat is generated. The separation is on the order of0.015 inch, so this non-uniformity in the heat source is not seen at theother side of the brass plate. Uniformity of temperature of the slidemay also be achieved by modifying the heating traces so that theyproduce heat in a non-uniform manner. Heat is lost to the surroundingair at all surfaces including the edges of the slide 37 and edges ofbrass plate 60. But heat is generated only in the heater 64 on thebottom surface of the brass plate. If the heat generation is uniformover the area of the heater so there is constant flux into the bottom ofthe brass plate, the center of the top of the glass slide will besignificantly hotter than the edges where heat can dissipate faster.This phenomenon can be accommodated if the heater flux is less in thecenter so that the temperature uniformity on the top of the glass slidecan be enhanced. A finite element heat transfer program, Mechanica byParametric Technologies (Waltham, Mass.) may be employed to model theheat flow in the system. The heater flux is preferably set to be asshown in FIG. 12, where the central area 63 covers 40% of the total andgenerates 8.3% of the total energy while the outer zone area 61generates the balance. Therefore, since the central area receives lessheat, the temperature on the slide that is directly above the centralarea is slightly cooler than the temperature of the slide that isdirectly above the oblong ring. The variation in temperature over theuseful area of the glass slide (the area upon which tissue is placed,which generally excludes the extreme edges of the slide) is about 0.2°C., when the heater is profiled as described. The temperature gradientfrom the center of the glass slide to an edge of the useful area of theglass slide is as follows: gradually rising 0.2° C. then graduallyfalling 0.2° C. The top surface of a wetted glass slide, which is oneinch wide and three inches long, maintains a specified temperatureuniformly over its useful area which is defined as the area centered onthe width of the slide that is 0.75 inches wide, 1.6 inches long andstarts 0.25 inches from the end opposite the label. The uniformity goalis to maintain a set temperature to within plus or minus two Celsiusdegrees over the useful area for any set temperature from 37° C. to 95°C.

[0058] In lieu of the aforementioned heating elements, a Peltier-typethermal transducer could be employed that is capable of both heating andcooling by reversing the polarity through the transducer. Such a coolingcapability may have utility in connection with certain potential usesand applications of the present invention such as performing in-situ PCR(discussed hereinafter).

[0059] A temperature sensor 68 is also mounted within cavity 73 to theunderside of heater 64. Several different types of sensors could be usedsuch as thermistors, RTD's, or thermocouples. In a preferred embodimentof the present invention an integrated circuit sensor 68 providingdirect temperature to digital conversion is used such as sensor modelno.DS1721 available from Dallas Semiconductor (Dallas, Tex.). Thissensor uses the I²C protocol to digitally report the sensed temperature.Similar sensors are available from National Semiconductor (Santa Clara,Calif.). The sensor selected must be accurate, repeatable, and have lowthermal mass.

[0060] A housing 70, preferably constructed of an injection moldedplastic, is provided for removably clamping heater/sensor unit 58 tocarousel 34 and for supporting glass slides 37. As best viewed in FIG.9, housing 70 defines a generally rectangular cavity 71 into whichheater/sensor unit 58 may be inserted and held by a clamp. At bottom ofhousing 70 there is defined a recessed area 72 that receives acorresponding rim 74 defined along the terminal edge of skirt 62 to holdheater sensor unit 58 in place. To further seal cavity 71 from thevarious reagents used during operation of the apparatus, a downwardlyextending lip 76 can be provided to more firmly engage rim 74. A bulge78 may also be provided along the outer wall of skirt 62 for furthersealing. The outside dimension of this bulge is slightly larger than theinside dimension of the housing, causing an interference that preventsliquids from entering cavity 71 below the bulge between the skirt andthe housing.

[0061] The base of the housing defines a set of apertures 80 (FIG. 8) toreceive machine screws that engage aligned bores defined in thecarousel.

[0062] Glass slides 37 rest against plate 60 captured by four upwardlydepending posts 82 which are integrally mounted to housing 70. The postsextend half way up the thickness of the glass slide 37 as can be seen inFIG. 9 since the glass slide is 0.04 inch thick, the posts are 0.02 inchbelow the top surface of the glass slide. It is important that the postsdo project to the top surface of the slide in order to prevent surfacetension of the aqueous solution from drawing the solution off. A problemwith devices known in the art that vertically clamp against the top ofthe slide is that they have a tendency to wick fluid off of the slide bycapillary action. It should be appreciated that other means forcapturing or supporting the slides may be employed.

Control Electronics (Printed Circuit Board)

[0063] With reference to the block diagram shown in FIG. 13, controlelectronics 52 consists of an annular printed circuit board with thecomponents necessary to receive temperature setpoint information fromstaining apparatus microprocessor 44 via a serial digital protocol 57and using this information to maintain each heater 64 at its setpoint.Not shown are those components (e.g. resistors, capacitors, etc.)readily understood by one of skill in the art to be included. Thecontrol of the heaters may be performed in a variety of methods. In apreferred embodiment, the heaters may be individually controlled by anintegrated circuit driver or individual transistors 53 capable ofswitching the heater current on and off. Thus, the processor may controlthe duty cycle of the heaters, as described subsequently. In analternate embodiment, the amount of power to the heaters may beregulated by processor 55 so that the heaters may be performed at apercentage of total capacity (e.g., 50% of maximum heating power). In apreferred embodiment, an integrated circuit (UDQ2559) available fromAllegro Microsystems (Worcester, Mass.). Serial communication 57 withmicroprocessor 44 (via slip ring assembly 56) preferably uses the I²C(Inter Integrated Circuit) serial bus protocol developed by PhilipsLabs, Eindhoven, The Netherlands. Alternative protocols could also beemployed such as RS232D, RS422 or others. Control electronics 52functions to both monitor the sensors 68 and control the heaters 64.Central to control electronics 52 is a microprocessor 55 (which is incommunication with memory 51) or other digital circuitry of sufficientcapability to communicate with staining apparatus microprocessor 44 viathe I²C serial bus, monitor the heater temperature sensors 68 and powerthe heaters 64 when the slide temperature needs to be raised at aparticular time. An example of such a microprocessor is PIC16C64Aavailable from Microchip Technology Inc., Chandler, Ariz. The programmust control each heater in response to the heater temperature sensorwhen compared with the setpoint temperature (or target temperature)provided by the staining system microcontroller. (See FIG. 14). Themicroprocessor 55 determines, based on a look-up table of the setpointtemperatures 47 in memory 51, how to control the heaters. The setpointtemperatures in look-up table 47 are received from serial communication57 with microprocessor 44. The microprocessor 55 obtains the actualtemperatures from sensors 68, and thereafter modifies the control of theheaters 64 based on the difference in actual temperature and setpointtemperature. This control of the heaters may be strictly on-off (i.e.,turn the heater on if its sensor temperature is below setpoint, and turnthe heater off if its sensor temperature is above setpoint) or it mayuse proportional, integral, and/or derivative control system algorithmsto provide a more controlled and accurate response.

[0064] The power distribution and controlling feedback system must besufficient so that the thermal platform can be precisely regulated tomimic the features in thermocycling technology (e.g. in-situ PCR) and becapable of rapid temperature ramp-ups and cool-downs (e.g. from 37° C.to 95° C. in under 3 minutes and cool over the same range in under 5min.). These features are particularly important for successful ISHstaining.

[0065] The processor controls the temperature of the slides by modifyingthe heat to the slides in order for the slides' temperature to be at thetarget temperature. In a preferred embodiment, modifying the heat to theslides is accomplished by modifying the output of the heaters. Referringto FIG. 14, there is shown a flow chart of the control of the heating ofthe individual slides. As shown at block 84, the target heatingtemperature (or setpoint temperature) for the individual slide isobtained. In the preferred embodiment, the target temperature is sentfrom microprocessor 42 to the control electronics 52. The targettemperature may alternatively be input from the operator or be readthrough the barcode on the slide. As shown at block 86, it is determinedwhether the temperature of the slide, as indicated by temperature sensor68, is above the target temperature. If yes, the heater is turned off,as shown at block 88. Alternatively, depending on the disparity betweenthe actual temperature and the target temperature, the amount of heatgenerated by the heater may be modified. For example, the duty cycle forthe heater may be reduced. Through pulse width modulation, the dutycycle for the heater may be modified (e.g., the duty cycle for theheater may be reduced from 50% (where the heater is on 50% of the time)to 25% duty cycle (where the heater is on 25% of the time)). In anotheralternative embodiment, the cooler can be turned on after the heater isturned off, if the actual temperature is significantly greater than thetarget temperature or if finer temperature control is sought.

[0066] In still another alternative embodiment, the processor maycontrol the amount of heat transferred to the slide by modifying theamount of heat transferred between the heater and the slide. As oneexample, the microprocessor may change the heat transmissioncharacteristics of a buffer between the heater and the slide, therebymodifying the amount of heat transferred to the slide, and therebymodifying the temperature of the slide.

[0067] If the temperature of the slide is less than the targettemperature, the heater is turned on, as shown at block 90. The controlof the heater may use proportional, integral, and/or derivative controlsystem algorithms to provide a more controlled and accurate response. Ina preferred embodiment, the amount of heat generated by the heater isbased on the disparity between the actual temperature and the targettemperature. For example, depending on the disparity in the temperaturebetween the current temperature and the target temperature, the heatermay be turned on for a 100%, 50%, etc. duty cycle. Thus, if the actualtemperature is greater than 2° C. away from the target temperature, theheater is turned on with a full duty cycle. As the actual temperatureapproaches the target temperature, the duty cycle of the heater isreduced thereby generating less total heat for the slide. In addition,once the heater achieves the target temperature, the processor 55maintains control by determining the difference between the actualtemperature and the target temperature, as shown at block 86. This loopis continued until the time for heating of the slide is over, as shownat block 92. Thus, if the slide is scheduled to be heated for apredetermined amount of time, the control of the temperature of theslide is performed until the predetermined amount of time has elapsed.

[0068] Moreover, based on the particular target temperature of a slide,it has been determined empirically the amount of heat necessary tomaintain the slide at the target temperature. For example, when theamount of heat is modified based on modifying the duty cycle of theheaters, the particular duty cycle for the heater has been determinedbased on the target temperature. These values are stored in look-uptable 47. Thus, as the actual temperature of the slide approaches thetarget temperature, the duty cycle is reduced to the empirical value inlook-up table 47. In this manner, the temperature of the slide may betransitioned from the current temperature to the target temperature.

[0069] Alternatively, the control of the temperature may be strictlyon-off (i.e., turn the heater on if its sensor temperature is belowtarget temperature, and turn the heater off if its sensor temperature isat or above the target temperature) or the control of the temperaturemay be proportional (i.e., modifying the amount of heat generated by theheater instead of the duty cycle so that the heater outputs a portion ofits total heating power, e.g. 50% of its total heater output).

[0070] Referring to FIG. 15, there is shown a flow chart of the controlof the cooling of the individual slides as an alternate embodiment ofthe invention. As shown at block 94, the target cooling temperature forthe individual slide is obtained. The target temperature is sent frommicroprocessor 42 to the control electronics 52. The target temperaturemay alternatively be input from the operator or be read through thebarcode on the slide. As shown at block 95, depending on the operationof the system, the slide may be cooled by the ambient air, oralternatively be cooled by cooling platforms or the like.

[0071] As shown at block 96, similar to FIG. 14, it is determinedwhether the temperature of the slide, as indicated by temperature sensor68, is below the target temperature. If yes, the cooler is turned off,as shown at block 97. Alternatively, depending on the disparity betweenthe actual temperature and the target temperature, the cooling amountgenerated by the cooler may be modified. For example, the duty cycle forthe cooler may be reduced. Through pulse width modulation, the dutycycle for the cooler may be modified.

[0072] If the temperature of the slide is greater than the targettemperature, the cooler is turned on, as shown at block 98. The controlof the cooler, similar to control of the heater, may use proportional,integral, and/or derivative control system algorithms to provide a morecontrolled and accurate response. In one embodiment, the cooling forcegenerated is based on the disparity between the actual temperature andthe target temperature. For example, depending on the disparity in thetemperature between the current temperature and the target temperature,the cooler may be turned on for a 100%, 50%, etc. duty cycle. Thus, ifthe actual temperature is greater than 2° C. away from the targettemperature, the cooler is turned on with a full duty cycle. As theactual temperature approaches the target temperature, the duty cycle ofthe cooler is reduced, thereby generating less total cooling energy forthe slide. In addition, once the cooler achieves the target temperature,the processor 55 maintains control by determining the difference betweenthe actual temperature and the target temperature, as shown at block 96.This loop is continued until the time for cooling of the slide is over,as shown at block 100.

Slip Ring Assembly

[0073] Slip ring assembly 56 uses technology employing rotating silverrings and silver graphite brushes. The slip ring must be of sufficientsize (about 3″ diameter) and capacity (about 10 amps per circuit) tocarry the I²C digital control signals (two circuits), power to the logic(two circuits), and power to the heaters (two circuits). Slip ringssuitable for this application are available from Fabricast, Inc, SouthEl Monte Calif., Airflyte Electronics, Bayonne, N.J., LittonPoly-Scientific, Blacksburg, Va. and others. A cable 57 operablyconnects the slip ring rotor to the control electronics 52 (FIG. 11). Astator bracket 59 is provided to mount the stator to apparatus 10 usingmachine screws or the like (mounting not shown). Referring to FIG. 16,there is shown a longitudinal section view of the slip ring assembly 56,with leads 49 which transmit data, logic power and heater power, asshown in FIGS. 5 and 16.

[0074] With reference to FIG. 3 the following components are mountedwithin apparatus 10 generally as described in U.S. patent applicationSer. No. 08/995,052: Liquid Coverslip™ dispense nozzle 37, stepper motor61, rinse dispense nozzles 36, fluid knife 38, bar-code reader 40, airvortex mixers 42, and wash volume adjust 39. The buffer heater asdisclosed in the above-referenced application has been removed. Bulkfluid module 22 (FIG. 2) is preferably constructed to accommodate aplurality of fluid receptacles as needed for ISH including SSC, DIwater, and cell conditioning buffers. The volume adjust nozzle 39 isconstructed to permit application of the plurality of fluids.

[0075] The invention herein also departs from the one disclosed in U.S.patent application Ser. No. 08/995,052 in the manner in which the washbuffer is wiped off the slide after being applied so as not to dilutethe next reagent applied. With the present invention nozzle 36 is aimedto spray fluid at the end of the slide from which the fluid is to beevacuated thereby causing evacuation of the fluid through capillaryaction.

Definitions

[0076] The following terms shall have the following meanings as usedherein:

[0077] “Tissue” means any collection of cells that can be mounted on astandard glass microscope slide including, without limitation, sectionsof organs, tumors sections, bodily fluids, smears, frozen sections,cytology preps, and cell lines.

[0078] “Targeted molecules” means detectable molecules found in cellsincluding without limitation nucleic acids, proteins, carbohydrates,lipids, and small molecules.

[0079] “Stain” means any biological or chemical entity which, whenapplied to targeted molecules in tissue, renders the moleculesdetectable under a microscope. Stains include without limitationdetectable nucleic acid probes, antibodies, and dyes.

[0080] “Treating” or “Treatment” shall mean application of a stain to atissue as well as other processes associated with such applicationincluding, without limitation, heating, cooling, washing, rinsing,drying, evaporation inhibition, deparaffinization, cell conditioning,mixing, incubating, and evaporation.

[0081] “Automated” or “Automatic” means activity substantially computeror machine driven and substantially free of human intervention.

Use and Operation

[0082] In operation, apparatus 10 may be used to perform in-situhybridization (ISH), in-situ PCR, immunohistochemistry (IHC), as well asa variety of chemical (non-biological) tissue staining techniques.Moreover, two or more of the above techniques may be employed during asingle run despite their differing temperature requirements due to theinventive heating system herein.

[0083] In-situ hybridization is clearly a technique that may beadvantageously employed with the present invention, either alone or incombination with other techniques, since many of the steps in ISH mustbe carefully temperature controlled for a precise period of time. Theprecise amount of heat for a specific period of time is necessary tosufficiently denature the DNA so that subsequent hybridization may occurwithout over-heating to the point where cell morphology is degraded.Different specimens may require different temperatures for denaturationdepending on how the tissue was prepared and fixed. The steps ofdenaturation, hybridization, and posthybridization washes each haveunique temperature requirements that depend on the particulars of theprobe and tissue being tested. These temperature requirements can becontrolled through the individualized control of the heaters, asdiscussed previously. DNA probes require and are typically hybridized atbetween 30°-55° C. while RNA probes are typically hybridized at highertemperatures with the time for hybridization varying from 30 min. to 24hours depending on target copy number, probe size and specimen type.Standard denaturation for cytogenetic preparations is performed at about72° C. for 2 min. while for tissue sections the conditions may vary from55° C. to 95° C. from 2 to 30 min. Post hybridization wash temperaturesmay vary from about 37° C. to 72° C. for 2 min. to 15 min. Saltconcentration may vary from 0.1× to 2× SSC. Probe detection temperaturesmay vary from ambient to 42° C. for 2 min. to 30 min.

[0084] The low mass of the plate 60 and heater 64 enables the rapidheating and cooling of the slide and consequently the tissue on theslide (i.e. from 37° C. to 95° C. in 180 seconds). The increasedrapidity of heating and cooling increases the efficiency of in situhybridization. The rapid annealing of the probe to the targetfacilitated by rapid temperature ramping increases the specificity ofthe probe. Concomitantly, the background is decreased and the quality ofthe resulting test is vastly improved. ISH may be employed to detectDNA, cDNA, and high copy mRNA. It can be applied to smears, tissue, celllines, and frozen sections. Typically, the specimen is mounted on a1″×3″ glass slide.

[0085] Hybridization or denaturation of DNA is absolutely essential tothe tissue staining process and requires that temperatures in the rangeof 92-100 degrees C. be quickly reached, precisely controlled andmaintained. The thermal platform brings treated tissue on microscopeslides to the required temperature range in less than 180 seconds withan accuracy of plus or minus 2 degrees C. Rapid loss of temperature inhybridized tissue is essential to successful staining and diagnosis. Afan or other rapid cooling feature may be added to bring the requiredtemperature to 37 degrees C. in less than 420 seconds.

[0086] The inventive apparatus permits the placement of multiple typesof specimens and ISH tests in the same run without compromising theunique requirements of each ISH test requirement (i.e., hybridizationtemperature 37-45° C. stringency, and wash concentrations). The systemmay run more than one detection chemistry in the same run on differentslides. As used herein “ISH” includes both fluorescent detection (FISH)and non-fluorescent detection (e.g. brightfield detection).

[0087] Apparatus 10 may also be employed for the simultaneousapplication of ISH and IHC staining to certain tissue sections to allowboth genetic and protein abnormalities to be viewed at the same time.This may be advantageous, for example, in assaying breast tumor sectionsfor both gene amplification and protein expression of HER-2/neu as bothhave been deemed to have clinical significance. See Ross et al. “TheHer-2/neu Oncogene in Breast Cancer,” The Oncologist 1998; 3:237-253.

[0088] The rapid heating and cooling by the thermal platform make thepresent invention amenable for use in in-situ PCR (polymerase chainreaction) which requires repeated cycles of higher and lowertemperatures. A limitation of PCR is the need to extract the target DNAor RNA prior to amplification that precludes correlation of themolecular results with the cytological or histological features of thesample. In situ PCR obviates that limitation by combining the celllocalizing ability of ISH with the extreme sensitivity of PCR. Thetechnique is described in U.S. Pat. No. 5,538,871 to Nuovo et al. whichis incorporated herein.

[0089] Sections embedded in paraffin require as a first stepdeparaffinization of the embedded tissue. Using the thermal platformeliminates the use of harsh chemicals such as xylene, through the use ofprecisely controlled heating of individual slides allowing the paraffinembedded in the tissue to melt out and float in aqueous solution whereit can be rinsed away. Paraffin, being less dense than water, onceliquified rises through the aqueous buffer on the tissue sample andfloats on top of this fluid. The liquid paraffin can then be removedfrom the microscope slide and away from the tissue sample by passing afluid stream, either liquid or gaseous, over the liquid paraffin.Details of this procedure are set forth in U.S. Patent ApplicationSerial No. 60/099,018 filed Sep. 3, 1998 which is incorporated herein. Asimilar technique may be employed to remove embedding materials otherthan paraffin such as plastics although the addition of etching reagentsmay be required.

[0090] Heating the tissue with thermal platforms 50 in an appropriateaqueous solution has been found to improve the accessibility of thestain to the target molecule in the cell (protein, nucleic acid,carbohydrate, lipid, or small molecule). Lack of accessibility may becaused by cross-linking of the molecules by aldehydes used in preservingthe tissue or by other changes in the confirmation caused by fixatives.Cross-linking of antigens causes a loss of antigenicity due to thechemical modification of antigenic proteins. This process of improvingaccessibility of the stain (biological or chemical) to the moleculartarget is referred to herein as “cell conditioning.” For DNA targets thepreferred conditioning solution is citrate buffer, the preferredtemperature is up to about 95 degrees, and the preferred time of heatingis about one hour. For protein targets the preferred conditioningsolution is citrate buffer and the preferred temperature is up to about100 degrees C. for about 42 minutes. Heating the tissue sample by thethermal platform decreases the degree of cross-linking in aldehydetreated tissue such that the modified antigen reverts to a formrecognizable by a corresponding antibody thereby enhancing the staining.For RNA targets the preferred conditioning solution is citrate bufferand the preferred temperature is up to about 75 degrees C. for about onehour. Many alternatives to citrate buffer may be employed as cellconditioning solution. A list of such solutions appears in AnalyticalMorphology, Gu, ed., Eaton Publishing Co. (1997) at pp. 1-40. Thesolutions should generally have known molarity, pH, and composition.Sodium dodecyl sulfate (SDS), ethylene glycol are perferably added tothe conditioning solution.

[0091] Typical In-Situ Hybridization (ISH), In-Situ PCR,Immunohistochemical (IHC), Histochemical (HC), or Enzymehistochemical(EHC) methods as carried out with the apparatus of this inventionincludes the following steps.

[0092] 1) Slides are prepared by applying a bar code to the slideindicating the In-Situ Hybridization, In-Situ PCR Immunohistochemical,Histochemical, or Enzymehistochemical process to be used with thesample.

[0093] 2) Inserting a batch of slides in the apparatus, mounting eachslide into a slide support.

[0094] 3) Closing the apparatus and beginning the treatment process.

[0095] 4) If the slides are to be deparaffinized in the apparatus as apretreatment, each slide will be dry heated to temperatures above 60° C.Following the dry heat, the slides are washed with about 7 ml of DIwater leaving a residual aqueous volume of about 300 μl. The slides arethen covered with about 600 μl of evaporation inhibiting liquid. Theslides remain at temperatures above 60° C. for an additional 6 minutesand are then rinsed again with about 7 ml DI water and covered with 600μl of evaporation inhibiting liquid. The temperature of lowered to 37°C. The slides are deparaffinized and ready for the next phase of theindicated process.

[0096] 5) Slides that are to be cell conditioned will be rinsed withabout 7 ml DI water. A volume-reducing fixture within the apparatus willlower the residual volume from about 300 μl to about 100 μl. Using avolume-adjusting fixture within the apparatus 200 μl of cellconditioning solution will be added to the slide. The slide will thenreceive about 600 μl of evaporation inhibiting liquid. The slidetemperature will be raised to the assigned temperature in a range of 37°C. to 100° C., and fluid cycling will commence and be repeated every 6-8minutes for a period of time up to 2 hours as set in the protocol.Slides are cooled to 37° C. and rinsed with ˜7 ml of APK wash solution.At this point the slides are ready for the next phase of the indicatedprocess.

[0097] 6) As each slide pauses in the reagent application zone, theappropriate reagent vessel is moved by the reagent carousel to thereagent application station. A metered volume of reagent is applied tothe slide. The reagent liquid passes through the evaporation inhibitingliquid layer to the underlying liquid layer.

[0098] 7) The slide carousel then proceeds, moving slides directly infront of vortex mixing stations. The vortex mixer jets stir the reagentson the slide surface below the evaporation inhibiting liquid layer.

[0099] 8a) For In-Situ Hybridization

[0100] If process requires protein digestion, slides are rinsed with ˜7ml of APK wash solution leaving a residual volume of ˜300 μL of buffer.The slide will then receive ˜600 μL of evaporation inhibiting liquid.Steps as described in steps 6 and 7 are repeated for digestive enzymeapplication. Selectable incubation times range from 2 min through to 32minutes at 37° C. The slides are rinsed with ˜7 mls of 2× SSC buffer,leaving a residual volume of ˜300 μL of buffer. A volume reducingfixture is used to shift the volume from ˜300 μL to ˜100 μL. Steps asdescribed in 6 and 7 are repeated for probe application. The slide willthen receive ˜600 μL of evaporation inhibiting liquid. Raise slidetemperature to specified temperature in a range of 37° C. to 95° C. fordenaturization or unfolding respectively of target and/or probe.

[0101] Selectable incubation times range from 2 min through to 18 hrs.

[0102] Rinsing occurs after hybridization employing user selectablestringency, which includes selectable salt concentrations of 2×, 1×,0.5×, 0.1× SSC and temperature range 37° C. to 75° C. Following theprobe step, the slides are washed with 1×APK wash buffer, and thenreceive ˜600 μL of evaporation inhibiting liquid. Probes are detecteddirectly as in the case of some labeled probes as in FISH, andindirectly for ISH using anti hapten antibody followed an appropriatedetection technology.

[0103] If Clearing is desired, following the detection steps for theprobe, slides will be rinsed with DI water and a detergent will beapplied to clear the slides of the evaporation inhibiting liquid. Againthe slides will be rinsed with DI water and the residual volume will beremoved with the use of the volume reducing fixture. The slides will bedry heated at temperatures at or above 37° C. until all aqueous isevaporated from the tissue, cells or smears.

[0104] 8b) For In-Situ PCR

[0105] If process requires protein digestion, slides are rinsed with ˜7ml of APK wash solution leaving a residual volume of ˜300 μL of buffer.The slide will then receive ˜600 μL of evaporation inhibiting liquid.Steps as described in steps 6 and 7 are repeated for digestive enzymeapplication. Selectable incubation times range from 2 min through to 32minutes at 37° C.

[0106] The slides are rinsed with ˜7 mls of DI Water, leaving a residualvolume of ˜300 μL of buffer. A volume reducing fixture is used to shiftthe volume from ˜300 μL to ˜100 μL.

[0107] Steps as described in step 7 are repeated for amplificationreagent application. Amplification reagents are formulated for deliveryto 100 μL residual slide volume at temperatures at or above 37° C. Theslide will then receive ˜600 μL of evaporation inhibiting liquid. Raiseslide temperature to specified temperature in a range of 37° C. to 95°C. for greater that 2 minutes to start PCR reaction. Heat cycling up to30 cycles, will commence from 55° C. for 1.5 minutes to 89° C. for 45seconds.

[0108] Following In-Situ PCR the slides will be subjected to In-SituHybridization as described in section

[0109] 8c) For IHC, HC, EHC protocols, the slides are rinsed with ˜7 mlsof 1×APK wash or appropriate buffer, leaving a residual volume of ˜300μL of buffer. A volume reducing fixture may or may not be used to shiftthe volume from ˜300 μL to ˜100 μL. The slide will then receive ˜600 μLof evaporation inhibiting liquid. Steps as described in step 7 arerepeated for antibody or other reagent application.

[0110] Selectable incubation times range from 2 min through to 32 min.

[0111] Selectable incubation temperatures range from 37° C. to 95° C.depending on whether cell conditioning or deparraffinization isrequired.

[0112] Throughout the procedure, the slides are washed with 1×APK washor appropriate buffer, and then receive ˜600 μL of evaporationinhibiting liquid. Proteins, carbohydrates, and enzymes are directlylabeled, as in fluorescence, or indirectly using an appropriatedetection technology.

[0113] At the conclusion of the designated staining procedures, theslides are prepared for coverslipping with the automated clearingprocedure, coverslipped, and reviewed microscopically for appropriatestaining, be it DNA/RNA, protein, carbohydrate or enzyme.

[0114] The procedures set forth above, including sequence of steps,application of reagents, and temperature parameters above are preferablypre-programmed into the host computer by the manufacturer. Certainparameters, such as the reaction time, may optionally be modifiable bythe user. Initial programming of the test is flexible enough to allowcomplex manipulation of the protocol and addition of multiple reagents(5-6 reagents) both before and after addition of the probe to the targettissue or specimen.

[0115] Within-run and between run temperature control is ±1% of thetarget temperature and may be controlled as described previously. Theoperator may run multiple complex ISH protocols in the same run. Thisincludes ability to program protocols for ISH methods that run at thesame denaturation temperatures. Likewise, the system is accessible forslide temperature calibration by the operator without tediousdismantling of the instrument. The protocol changes for user-defined ISHprotocols are protected by a security access. The system is barcodedriven for both the slide and reagent system. There is also the optionfor operator manual control of all major hardware functions includingreagent dispense, wash dispense, coverslip (high and low temperature)dispense, slide indexing and temperature control. This enables the userto help troubleshoot problems. User defined protocols allow the operatorto control the temperature of all phases of the reaction exceptdetection temperature. The software includes pre-programmed optimizedprotocols resident in the software to allow continuous introduction ofthe optimized turnkey probes.

EXAMPLES

[0116] The following non-limiting examples further illustrate and detailuses and applications of the invention disclosed herein.

Example I Detection of High Risk Strains of Human Papaloma Virus (HPV)Using Automated In-Situ Hybridization

[0117] A cervical smear, collected by standard collection devices suchas a cytobrush or by a ThinPrep™ slide method (Cytec, Inc.) which wouldnormally be stained by the Papanicolau stain (Pap smear), is subjectedto in situ hybridization with a probe set specific to High Risk HPVtypes. The specimen slide is loaded into the slide holder of theapparatus according to the present invention (hereinafter referred to as“staining apparatus”). The system is set to run the HPV in situ program.This program performs all the steps of the in situ reaction with nointeraction required from the user once the program has been started.

[0118] The staining apparatus first performs Slide Pretreatment: at roomtemperature 1×APK (10×APK Ventana P/N 250-042) rinse and Coverslip™application, then a Protease digestion for 4 minutes at 37° C. withProtease 1 followed by a rinse step in 2× SSC. Following the 2×SSC rinsethe residual slide volume is reduced from about 300 microliters to about100 microliters and Coverslip™ is applied. The FITC-label DNA probecocktail premixed with Hybridization Solution is applied to the slidefrom a Ventana definable dispenser. The specimen and the slide is heatedto 72° C. for 4 minutes to denature both the probe and specimen DNA. Thestaining apparatus then ramps the temperature of the slide down to 37°C. and hybridization occurs for 2 hours at 37° C. The staining apparatusremoves the probe mixture and Coverslip™ from the slide and performs thePost-Hybridization Washes. Post-Hybridization Wash solution (2×SSC) andCoverslips are added to the slide and the instrument heats the slide to45° C. for 10 minutes. The staining apparatus removes thePost-Hybridization Wash solution and begins the Detection steps. First,the staining apparatus rinses the slides with 1×APK and appliesCoverslip. Then, an anti-FITC (serotec P/N MCA1320) antibody is appliedfrom a dispenser to the slide and incubates the slide for 20 minuteswhile heating the slide to 37 degrees C. followed by APK rinse andCoverslip application. Then, a biotin labeled secondary antibody isadded and incubated for 8 minutes at 37° C., also followed by an APKrinse and Coverslip application. A Streptavidin-Alkaline Phosphatesconjugate is placed on the slide and staining apparatus incubates theslide for 30 minutes at 37 degrees C. After an APK rinse, Ventana BlueDetection Reagents are added to the slide and incubated for 20 minutesat room temperature. Ventana Blue Kit (P/N 760-060) comprises of Biotinlabeled secondary antibody, Streptavidin-Alkaline Phosphatase andNBT/BCIP substrate. The slide is rinsed with DI Water, and the slide isheat dried by the instrument. At this Point, Nuclear Fast Redcounterstain is applied to the slide, incubated for 5 minutes at roomtemperature, and the slide is rinsed with water.

Example II Detection of mRNA of Epstien Barr Virus (EBER) in ParaffinEmbedded Tissue Using Automated In-Situ Hybridization

[0119] A 5 micron section was cut of spleen # EBV 37A and placed on asupper frost glass staining slide. The specimen slide was loaded intothe slide holder of the apparatus according to the present invention(hereinafter referred to as “staining apparatus”). The stainingapparatus was programmed to run the EBER in situ program. This programwill perform all the steps of the in situ reaction with no interactionrequired from the user once the program has been started.

[0120] The staining apparatus first performs deparaffinization: slideswere dry heated to 65° C. for 6 minutes, room temperature DI waterrinses the slide, leaving 300 μL residual volume and 600 μL liquidcoverslip, which prevents evaporation and protects the slide specimenfrom drying. The temperature remains at 65° C. to melt away theparaffin. Cell conditioning was done by rinsing the slide with DI water,then reducing the residual volume, and applying 200 μL cell conditioningbuffer (Citrate Buffer) and 600 μL liquid Coverslip™. The slide washeated to 75° C. and the conditioning buffer and Coverslip™ werereapplied every 8 minutes for 40 minutes. The slide temperature wascooled to 37° C. and the slide was rinsed with room temperature 1×APKWash (Ventana 10×APK P/N 250-042) leaving ≈300 μL residual slide volumeand ≈600 μL of liquid coverslip was applied. Protease digestion was for8 minutes with Protease 1 (Ventana P/N 250-2018) at 37° C. Followingdigestion the slide was rinsed with room temperature 2× SSC (20× SSCVentana P/N 650-012). Using a volume-reducing fixture in the stainingapparatus the residual side volume was reduced from ≈300 μl to ≈100 μLand ≈600 μL of liquid coverslip was applied to the slide. TheDig-labeled (Boehringer Mannheim cat # 1 417 231) oligonucleotide probewhich was designed to target mRNA for EBER was premixed withHybridization solution and the reagent was placed into a Ventana userdefined dispenser (Ventana P/N 551-761). The probe was applied to thespecimen and the slide was heated to 75° C. for 4 minutes to unwind theoligonucleotide and specimen mRNA. The staining apparatus then ramps toa temperature of the slide to 37° C., to hybridize for 2 hours. Thestaining apparatus removed the probe solution and Coverslip™ from theslide and performed 3 Post-Hybridization Washes. The Post HybridizationWashes consisted of washing the slide with 2× SSC at 42° C. for 4minutes then washing the slide with 1× SSC at 42° C. for 4 minutes andfinally 0.5× SSC 42° C. for 4 minutes. The staining apparatus thenperformed the detection steps. The slide was washed with 1× APK andcoverslip was applied. An anti-Dig antibody (Sigma P/N D-8156) wasapplied to the slide and incubated for 16 minutes at 37° C. followed by1×APK wash and Coverslip™ application. Then, a biotin labeled secondaryantibody was added to the slide and incubated for 8 minutes at 37° C.,followed by 1×APK wash and coverslip application. Following thesecondary antibody, Streptavidin-Alkaline Phosphatase conjugate wasapplied and incubated for 30 minutes at 37° C. After an APK wash andcoverslip application Ventana Blue Detection Reagents were applied tothe specimen and incubated for 20 minutes at 37° C. The slide was thenwashed with water and heat dried by the instrument. The biotin labeledsecondary antibody, Streptavidin-Alkaline Phosphatase, and DetectionReagents are components of Ventana Blue Kit (Ventana P/N 760-060).Following dehydration of the specimen, the slide was covered with aglass coverslip and reviewed microscopically.

Example III On-Slide PCR Amplification

[0121] The polymerase chain reaction (PCR) is a technique that permitsthe amplification and detection of a target nucleic acid molecule. Thistechnique has a wide variety of biological applications, including forexample, DNA sequence analysis, probe generation, cloning of nucleicacid sequences, site-directed mutagenesis, detection of geneticmutations, diagnoses of viral infections, molecular “fingerprinting,”and the monitoring of contaminating microorganisms in biological fluidsand other sources.

[0122] In PCR, an exponential amplification of the target molecule isaccomplished by subjecting the target molecule to repeated rounds, orcycles, of denaturation, annealing, and polymerase-mediated extension.The step of denaturing of the target molecules in the tissue sample isperformed by independently controlling the temperature of the heaters asdiscussed above. The step of annealing is performed in the presence of amolar excess of at least two oligonucleotide primers, wherein at leastone primer corresponds to the target molecule sequence and at least oneother primer corresponds to a sequence that is complementary to thetarget molecule. The step of annealing at least two oligonucleotideprimers to the target molecules is performed by independentlycontrolling the temperature of the heaters. The step of extension isperformed in the presence of a polymerase, most preferably athermally-stable DNA polymerase. The polymerase-mediated extension onthe annealed oligonucleotide primer-target molecules is performed byindependently controlling the temperature of the heaters. The steps ofdenaturing, annealing and performing polymerase-mediated extension isrepeated at least one time, and in a preferred embodiment, apredetermined number of times (e.g., 25).

[0123] The feasibility of using the apparatus of the present inventionto perform on-slide PCR amplification of a target molecule was examinedusing a 500 bp murine PS2 cDNA template (Lefebvre et al., 1993, J. CellBiol. 122:191-98) subcloned into the pBluescript+vector (Stratagene, LaJolla, Calif.). An amplification premix was first prepared, the premixconsisting of 20 ng of the PS2 plasmid template, 100 pmol each of the T3(5′-A-A-T-T-A-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-3′: SEQ ID NO: 1;Stratagene) and T7 (5′-G-T-A-A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G-G-G-C-3′;SEQ ID NO: 2; Stratagene) oligonucleotide primers, 5 μL of a dNTP mix,20 μL of 10× reaction buffer (Stratagene), 2 μL of 5% Brij35, 78 μL 0.5%Foam Blast® 106 (ROSS Chem, Inc. Fountain Inn, S.C.), and 5U of Taqpolymerase (Stratagene), in a total volume of 128 μL. Two types of dNTPmix were used in the various PCR amplifications performed using theapparatus of the present invention. The first dNTP mix, which wasemployed for standard PCR amplifications, consisted of 10 μL each ofdCTP, dGTP, dATP, and dTTP (100 mM deoxyribonucleotide stocks;Stratagene), in a total volume of 100 μL. The second dNTP mix(hapten-dNTP mix), which was employed for hapten incorporation duringPCR amplification, consisted of 10 μL each of dCTP, dGTP, and dATP, 9.4μL dTTP, and 2.4 μL of either Digoxigenin-11-dUTP (Roche, Indianapolis,Ind.) or Biotin-16-dUTP (Roche), in a total volume of 100 μL

[0124] For on-slide PCR amplification, the PCR premix was applied onto aslide and then covered with 90 μL of EZ PREP™ (Ventana Medical Systems,Inc., Tucson, Ariz.) and LIQUID COVERSLIP™ (Ventana Medical Systems,Inc., Tucson, Ariz.). For standard PCR amplification using a thermalcycler, the PCR premix was placed into a PCR tube containing 90 μL EZPREP™ and the reaction mix was covered with 100 μL of mineral oil.On-slide PCR amplification was performed at 94° C. for 2 minutes, 50° C.for 4 minutes, and 70° C. for 2 minutes for 25 cycles. The step ofdenaturing, in a preferred embodiment, is performed by controlling theheaters at 94° C. The step of annealing, in one embodiment, is performedby controlling the heaters to between 37° C. and 65° C., and in apreferred embodiment to 50° C. The step of performingpolymerase-mediated extension, in one embodiment, is performed bycontrolling the heaters to between 65° C. and 75° C., and in a preferredembodiment to 70° C. Additional EZ PREP™ and LIQUID COVERSLIP™ wasdispensed onto the slide following the fifth, tenth, fifteenth, andtwentieth cycles. Standard PCR amplification was performed at 94° C. for30 seconds, 50° C. for 45 seconds, and 70° C. for 1 minute for 25cycles.

[0125] For gel electrophoretic analysis of on-slide PCR amplification,the on-slide PCR reaction mix was collected and centrifuged in a 50 mltube. Portions of the on-slide and standard PCR amplifications were thenanalyzed on a 1.8% agarose gel. FIG. 17 illustrates that the expectedPCR amplification product was generated in both the on-slide (lanes 3and 4) and standard (lanes 1 and 2) PCR amplifications for PCRamplifications using the standard dNTP mix (lanes 2 and 4) or ahapten-dNTP mix (lanes 1 and 3).

Example IV On-Slide Detection of On-Slide PCR Amplification

[0126] The feasibility of on-slide detection of on-slide PCRamplification using the apparatus of the present invention wasdemonstrated using human beta-actin and human histone cDNA templatessubcloned into the pBluescript+vector. An amplification premix,comprising the hapten-dNTP mix, was prepared essentially as described inExample III.

[0127] For on-slide PCR amplification, the PCR premix was applied onto aslide with 90 μL of EZ PREP™ and then the reaction mix was covered withLIQUID COVERSLIP™. The apparatus of the present invention was configuredto perform both on-slide PCR amplification and on-slide hybridization.On-slide PCR amplification was performed as described in Example III.Following on-slide PCR amplification, on-slide antibody hybridizationwas performed as follows: 100 μL of RiboHybe buffer (Ventana MedicalSystems, Inc., Tucson, Ariz.) was dispensed onto the slide; the slidewas incubated at 37° C. for 6 minutes, 95° C. for 6 minutes; and 37° C.for 6 hours; the slide was rinsed once with 1× SSPE at 37° C. for 4minutes and once with 0.1× SSPE at 37° C. for 4 minutes; one drop of a1/200 dilution of murine anti-digoxin antibody (Sigma, St. Louis, Mo.)was dispensed onto the slide and the slide was incubated at 37° C. for20 minutes; one drop of a 1/200 dilution of rabbit anti-mouse IgG-TRITClabeled antibody (Sigma) was dispensed onto the slide and the slide wasincubated at 37° C. for 20 minutes; and one drop of a 1/200 dilution ofgoat anti-rabbit IgG-TRITC labeled antibody (Sigma) was dispensed ontothe slide and the slide was incubated at 37° C. for 20 minutes.

[0128] For on-slide antibody hybridization, the slides were rinsed withdH₂O, rinsed in ethanol, and then scanned using a GenePix 4000microarray scanner (Axon Instruments, Inc., Foster City, Calif.). FIGS.18A-18B illustrate the results obtained following on-slide PCRamplification and on-slide antibody hybridization. Prior to PCRamplification, template cDNA was spotted onto poly-lysine coated slidesin 6×3 spot sets. The human histone cDNA template was spotted in areasmarked as A, C, D, H, J, L, T, V, and X (see FIG. 17B); the humanbeta-actin cDNA template was spotted in areas marked as G, I, K, M, O,Q, S, U, and W; and a control plant cDNA template was spotted in areasmarked as B, D, N, P, and R. FIGS. 18A-18B demonstrate the specificityof on-slide PCR amplification and antibody hybridization, since theTRITC signal was detected only for the human cDNA templates and not forthe plant cDNA template.

[0129] Although certain presently preferred embodiments of the inventionhave been described herein, it will be apparent to those skilled in theart to which the invention pertains that variations and modifications ofthe described embodiment may be made without departing from the spiritand scope of the invention. Accordingly, it is intended that theinvention be limited only to the extent required by the appended claimsand the applicable rules of law.

What is claimed is:
 1. Apparatus for analysis of biological materialscomprising: a plurality of heating devices, each heating device adaptedto receive a slide and each heating device including a heater and asensor; heating device support, the heating devices being on the heatingdevice support; and control electronics in communication with theheating devices for receiving data from the sensors of the heatingdevices and for individually controlling the heaters of each of theheating devices.
 2. A method for amplifying a target molecule withintissue samples mounted on slides for a biological apparatus of claim 1comprising the steps of: denaturing target molecules in the tissuesample by independently controlling the temperature of the heaters;annealing at least two oligonucleotide primers to the target moleculesby independently controlling the temperature of the heaters; performingpolymerase-mediated extension on the annealed oligonucleotideprimer-target molecules by independently controlling the temperature ofthe heaters; and repeating the steps of denaturing, annealing andperforming polymerase-mediated extension at least one time.
 3. Themethod of claim 2 wherein the step of repeating is performed apredetermined number of times.
 4. The method of claim 2 wherein the stepof denaturing includes controlling the heaters so that the temperatureis at least 94° C.
 5. The method of claim 2 wherein the step ofannealing includes controlling the heaters so that the temperature isbetween 37° C. and 65° C.
 6. The method of claim 2 wherein the step ofannealing includes controlling the heaters so that the temperature isapproximately 50° C.
 7. The method of claim 2 wherein the step ofperforming polymerase-mediated extension includes controlling theheaters so that the temperature is between 65° C. and 75° C.
 8. Themethod of claim 2 wherein the step of performing polymerase-mediatedextension includes controlling the heaters so that the temperature isapproximately 70° C.